Ionizing radiation (IR) remains one of the mainstays of cancer therapy. The most deleterious lesion induced by IR is the DNA double-strand break (DSB). Accurate repair of DSBs is essential for preventing loss of genomic integrity and malignant transformation. Efficient repair of DSBs also underlies the resistance of many cancers to radiation therapy. A cell must choose between two major repair pathways to fix these breaks - non- homologous end joining (NHEJ), an error-prone pathway that is operative in all phases of the cell cycle or homologous recombination (HR), an error-free pathway that is restricted to the post-replicative phases of the cell cycle. Optimal usage of these two pathways is vital for the maintenance of genomic integrity and cell survival in the face of genomic insults. The DNA end resection step of HR is a pivotal point at which correct repair pathway choice is exercised. Importantly, research from our lab and others has established that the 5' to 3' exonuclease EXO1 is a critical player in DNA end resection and repair pathway choice in human cells. While DNA end resection is currently an avidly researched topic in the field of DNA repair, the exact sequence of molecular events involving EXO1 that allows commitment to a particular repair pathway is not well worked out. Exciting new results from our lab demonstrate that EXO1 is phosphorylated by CDKs 1/2 in a cell cycle- dependent manner and by ATM/ATR in a DNA damage-dependent manner to promote DNA end resection. However, soon after DNA damage, EXO1 is SUMOylated, ubiquitinated, and rapidly targeted for degradation, presumably to prevent uncontrolled resection of DNA ends. It is important to mechanistically understand how these and other post translational modifications stimulate or restrain EXO1's functions in the cellular response to IR. Towards this goal, we propose to develop a comprehensive picture of cell cycle- and IR-dependent modifications and interacting partners of EXO1, and to mechanistically understand how these modifications promote EXO1 activation and subsequent degradation in response to IR. Based upon our preliminary results, we hypothesize that EXO1 activation and inactivation is a tightly controlled process involving phosphorylation, SUMOylation and ubiquitination events that fine-tune DNA end resection, optimize DSB repair, and preserve genomic integrity. Understanding the sequence and functions of EXO1 post- translational modifications and the exact choreography of EXO1 and its interacting partners at DSBs will be of paramount importance in developing more effective radiosensitization approaches that target the critical DNA end resection step. Specifically, we propose to: 1) Test the hypothesis that phosphorylation of EXO1 by CDKs and PI3KKs regulates DNA end resection and influences repair pathway choice, 2) Test the hypothesis that EXO1 degradation post-radiation restrains DNA end resection and preserves genomic integrity, and 3) Test the hypothesis that blocking EXO1 activation with CDK 1/2 inhibitors may be a viable strategy for therapeutically sensitizing cancers to ionizing radiation.